In vitro effect of visfatin on endocrine functions of the porcine corpus luteum

Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to examine the visfatin effect on the secretion of steroid (P4, E2) and prostaglandin (PGE2, PGF2α), the mRNA and protein abundance of steroidogenic markers (STAR, CYP11A1, HSD3B, CYP19A1), prostaglandin receptors (PTGER2, PTGFR), insulin receptor (INSR), and activity of kinases (MAPK/ERK1/2, AKT, AMPK) in the porcine corpus luteum. We noted that the visfatin effect strongly depends on the phase of the estrous cycle: on days 2–3 and 14–16 it reduced P4, while on days 10–12 it stimulated P4. Visfatin increased secretion of E2 on days 2–3, PGE2 on days 2–3 and 10–12, reduced PGF2α release on days 14–16, as well as stimulated the expression of steroidogenic markers on days 10–12 of the estrous cycle. Moreover, visfatin elevated PTGER mRNA expression and decreased its protein level, while we noted the opposite changes for PTGFR. Additionally, visfatin activated ERK1/2, AKT, and AMPK, while reduced INSR phosphorylation. Interestingly, after inhibition of INSR and signalling pathways visfatin action was abolished. These findings suggest a regulatory role of visfatin in the porcine corpus luteum.

The corpus luteum (CL) is a transient gland that is formed in the mammalian ovary, and its proper functioning determines the maintenance of pregnancy as well as the cyclicity of the ovary.The CL performs its function mainly by secreting steroid hormones.The most important of them, progesterone (P 4 ), prepares the uterine wall for embryo implantation and prevents its contractions and further rejection of the fetus 1 .Estradiol (E 2 ), although secreted in smaller amounts, affects the generation of the CL by the formation of blood vessels 2 .In the process of steroidogenesis, in addition to the steroidogenic acute regulatory protein (STAR), which transports cholesterol from the outer to the inner mitochondrial membrane, two main types of enzymes are involved: cytochromes P450 and steroid oxidoreductases.Cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B) are enzymes involved in the synthesis of P 4 , while cytochrome P450 family 19 subfamily A member 1 (CYP19A1) is responsible for the conversion of androgens to estrogens, including E 2 3 .In addition, the CL synthesizes and secretes prostaglandins such as prostaglandin E 2 (PGE 2 ) and prostaglandin F 2α (PGF 2α ), which act through specific receptors: PTGER isoforms and PTGFR and control luteinization and luteolysis, respectively 4 .Together, the aforementioned hormones create a unique environment for the proper functioning of the CL.Recent studies also point to other important factors connected with the development, lifespan, and regression of the CL, including adipokines 5 .Most of them have a luteotropic effect: apelin stimulates P 4 secretion by increasing HSD3B expression 6 , and vaspin upregulates P 4 and E 2 synthesis and increases the ratio of PGE 2 to PGF 2α secretion in the porcine CL 7 .In contrast, adiponectin causes a decrease in P 4 secretion by porcine luteal cells isolated in the mid-luteal phase of the cycle 8 .Disturbances in the luteal endocrine function lead to numerous negative consequences, primarily luteal phase deficiency, defined as decreased production of P 4 , both in quantity and duration.Luteal phase dysfunction may result in premature regression of the gland and subsequent transition to an infertile cycle 9 .Understanding the mechanism of steroidogenesis during the

The effect of visfatin on basal and LH-and INS-induced P 4 and E 2 secretion by porcine luteal cells during the estrous cycle
A two-way analysis of variance (ANOVA) demonstrated that the secretion of P 4 and E 2 was affected by the visfatin, FK866, and the interaction of those factors.However, the effect did not occur in all cases.The impact of studied factors depended not only on the studied hormone, the treatment that stimulated steroid hormone secretion, but also on the phase of the estrous cycle.Detailed results of the two-way ANOVA are provided in Supplementary Tables 1 and 2.
Moreover, post-hoc testing (Tukey's post-hoc test) demonstrated that on days 2-3 of the estrous cycle, visfatin (10 and 100 ng/mL) decreased the P 4 concentration.We also noted that LH, INS, and LH with INS upregulated the P 4 level, while the addition of visfatin caused suppression of P 4 secretion to the control level.Interestingly, we observed no differences between the effects of LH together with INS on P 4 levels and these factors added separately (p < 0.05, Fig. 1A).In the presence of FK866, visfatin (10, 100 ng/mL) enhanced P 4 release.Interestingly, FK866 did not change the effect of visfatin in combination with LH or INS, but abolished the influence of visfatin added together with LH and INS compared to the effects of these treatments without a blocker (p < 0.05, Fig. 1B).
On days 10-12 of the estrous cycle, visfatin (10 and 100 ng/mL) increased P 4 release.INS alone did not affect P 4 level, while LH alone and with INS increased P 4 secretion.Moreover, we observed the additive effect of visfatin and LH: it led to highest the P 4 level (p < 0.05, Fig. 1C).In the presence of FK866, we noticed the inhibitory effect of visfatin added with LH and LH with INS as well as suppression of the action of visfatin added alone at the dose of 10 and 100 ng/mL (p < 0.05, Fig. 1D).
In the late luteal phase, on days 14-16 of the estrous cycle, all tested visfatin doses downregulated the P 4 level.LH added alone and with INS increased P 4 secretion.Moreover, the addition of visfatin resulted in a decrease in the P 4 level compared with the action of LH and INS together.Similarly, visfatin administered together with INS lowered the P 4 level compared with the control and INS, which alone did not affect P 4 level (p < 0.05, Fig. 1E).However, in the presence of FK866, only the effect of visfatin added alone at the concentration of 1 ng/mL was abolished.Also, in the presence of FK866, we noted the stimulation of P 4 release as a result of the combined administration of visfatin with LH but no effect on the suppression of P 4 by visfatin in the presence of INS (p < 0.05, Fig. 1F).
The E 2 level was higher after the treatment with visfatin (10 and 100 ng/mL) on days 2-3 of the estrous cycle.Similarly, LH and/or INS stimulated E 2 secretion, and the addition of visfatin with these hormones also increased E 2 level (p < 0.05, Fig. 2A).FK866 abolished the basal visfatin effect on E 2 secretion at doses of 10 and 100 ng/mL as well as the stimulatory influence of visfatin added together with LH and LH with INS but not with INS alone.The E 2 levels that were stimulated by combined LH and INS treatment with visfatin were significantly lower in presence of FK866 (p < 0.05, Fig. 2B).
On days 10-12 of the estrous cycle, E 2 secretion was higher after treatment with visfatin at the dose 10 ng/ mL, and decreased when visfatin was added at 1 ng/mL.LH and INS stimulated E 2 release but visfatin enhanced this effect only when administered with LH (p < 0.05, Fig. 2C).The use of FK866 alone did not affect E 2 secretion, but it reversed the inhibitory effect of visfatin at a dose of 1 ng/mL.The visfatin at doses of 1, 10 and 100 ng/ mL significantly stimulated E 2 secretion in the presence of FK866.Moreover, we noted that the effect of visfatin together with LH was also abolished in the presence of FK866 (p < 0.05, Fig. 2D).
On days 14-16 of the estrous cycle, visfatin did not affect the E 2 concentration, except when it was administered together with INS, where it increased E 2 level compared with the control and INS alone (p < 0.05, Fig. 2E).Interestingly, FK866 and visfatin at all tested doses increased E 2 secretion in this phase of the estrous cycle.Additionally, we observed that in the presence of FK866, visfatin together with LH and LH with INS increased E 2 levels, which we did not observe without the addition of the blocker.FK866 did not change the effect of visfatin in combination with INS (p < 0.05, Fig. 2F).

The effect of visfatin on the mRNA and protein abundance of steroidogenic markers in porcine luteal cells
We noted that visfatin dependently on the dose used, increased mRNA levels of steroidogenic markers: at the concentration of 10 ng/mL STAR (p < 0.05, Fig. 3A), at 1 and 10 ng/mL CYP11A1 and HSD3B (p < 0.05, Fig. 3C,E), while at all tested doses CYP19A1 (p < 0.05, Fig. 3G).Visfatin decreased CYP11A1 mRNA level at dose of 100 ng/mL (p < 0.05, Fig. 3C).In addition, all tested visfatin doses stimulated the protein abundance of STAR, CYP11A1, and HSD3B (p < 0.05, Fig. 3B,D,F).However, CYP19A1 protein expression was decreased after visfatin treatment at the dose of 1 ng/mL but increased at 100 ng/mL (p < 0.05, Fig. 3H).Overall, FK866  did not change the expression of studied markers except for CYP11A1 protein (p < 0.05, Fig. 3D) and CYP19A1 mRNA (p < 0.05, Fig. 3G), which levels were reduced compared with the control.The addition of the FK866 together with visfatin at the dose of 10 ng/mL resulted in the abolition of the stimulatory effect of visfatin on the mRNA and protein abundance of all examined steroidogenic markers, except for STAR mRNA level as well as for CYP19A1 protein levels where treatment with visfatin at this dose and FK866 increased the protein concentrations (p < 0.05, Fig. 3A,H).

The effect of visfatin on prostaglandin secretion by porcine luteal cells during the estrous cycle
Secretion of luteotropic PGE 2 was significantly higher after treatment with visfatin at the doses of 10 and 100 ng/ mL on days 2-3 and 10-12 (p < 0.05, Fig. 4A,B) of the estrous cycle, while there was no effect on days 14-16 (Fig. 4C).Furthermore, treatment of the cells with visfatin and FK866 abolished the visfatin effect on the PGE 2 secretion on days 2-3 and 10-12 of the estrous cycle (p < 0.05, Fig. 4A,B), whereas on days 14-16 reduced PGE 2 secretion compared with control and visfatin (10 ng/mL) groups (p < 0.05, Fig. 4C).

The effect of visfatin on the expression of prostaglandin receptors in porcine luteal cells
Expression of PTGER2 mRNA was upregulated by visfatin at the dose of 1 and 10 ng/mL (p < 0.05, Fig. 5A), while PTGER2 protein abundance was reduced by all visfatin doses (p < 0.05, Fig. 5B).FK866 abolished the effect of visfatin on the PTGER2 mRNA level (p < 0.05, Fig. 5A), but reduced PTGER2 protein abundance relative to visfatin (10 ng/mL) and control groups (p < 0.05, Fig. 5B).

Discussion
The CL is a very dynamic structure; it is formed after ovulation from the Gc and theca cells, which undergo luteinization in just a few days.This is followed by a period of increased P 4 secretion, aiming to prepare the uterus for embryo implantation.However, in the absence of fertilization, the CL must undergo regression to allow a new cycle to begin.This ensures the proper cyclicity of the ovary 1 .Adipokines are recognized as factors that regulate luteal tissue function in livestock and are identified as mediators linking energy balance and fertility 30 .Consequently, adipokines have been extensively studied within the female reproductive system.One of them, visfatin, has been widely demonstrated to affect steroidogenesis in ovarian follicles in humans 18 , mice 20 , cattle 22,23 , and hens 21 .So far, there has been no attempt to determine its impact on steroid or prostaglandin secretion by porcine luteal cells.We addressed this lacuna and comprehensively demonstrated for the first time that visfatin is an important regulator of hormone secretion in the porcine CL throughout the entire luteal phase, during which hormone secretion undergoes dynamic changes.Based on our findings, we noted the modulatory (stimulatory or inhibitory) effect of visfatin on luteal steroidogenesis dependent on the phase of estrous cycle.On days 2-3 and 14-16 of the estrous cycle, when P 4 production is lower 31 , visfatin downregulated P 4 secretion.Furthermore, after administration with LH and/or INS, visfatin also reduced P 4 secretion in the early luteal phase, similarly to treatment together with INS and LH with INS in the late luteal phase.Therefore, it seems that visfatin acts as a balancing factor, preventing premature or prolonged excessive P 4 secretion.In the mid-luteal phase, during the heightened activity of luteal cells 31 , there was an increase in the secretion of this steroid under the influence of visfatin.Moreover, the action of LH with visfatin is additive, leading to high P 4 production reaching the level of about 100 ng/mL.Our previous studies indicated high expression of visfatin in the middle CL, and we observed that P 4 itself stimulated visfatin protein expression and secretion by porcine luteal cells in the early and middle luteal phase, suggesting a role for visfatin in CL function 24 .Moreover, we noted stimulation of E 2 secretion by visfatin alone on days 2-3 and 10-12 of the estrous cycle, and the lack of an effect at the end of the luteal phase.However, visfatin in combination with LH and INS increased the release of E 2 at the end of the luteal phase as well as with INS on days 2-3 of the estrous cycle.Because E 2 , and more precisely its metabolites, have a beneficial effect on angiogenesis in the CL 2 , which has also been described for other tissues 32 , it may suggest a positive influence of visfatin on the development of the CL.
Our results are in good agreement with the published studies of visfatin's action on ovarian steroidogenesis.In the CL, the effect of visfatin on steroid secretion has only been determined in water buffalo.That study indicates the stimulatory effect of this adipokine on the P 4 secretion together with increased mRNA level of STAR , CYP11A1, and HSD3B, which are primarily involved in P 4 synthesis 23 .In Gc, the visfatin effect depends on the species, the maturity of the animals, and the presence of other stimulators of steroidogenesis, such as insulin growth factor 1 (IGF-1) or follicle-stimulating hormone (FSH).For example, in human primary Gc and the  www.nature.com/scientificreports/KGN cell line, visfatin increased IGF-1-induced P 4 and E 2 secretion, while there were no changes after administration with FSH 18 .Similarly, in water buffalo, visfatin at a dose of 10 ng/mL and also in the presence of IGF-1 stimulated E 2 secretion and the gene expression of CYP19A1 23 .Subsequently, visfatin increased the release of P 4 and E 2 , which was associated with an increase in STAR and HSD3B protein in cultured bovine Gc 22 .On the other hand, there are reports on the downregulation of steroidogenesis in hens, and mice.Diot et al. 21reported that in hens treatment of Gc with visfatin halved basal and IGF1-induced P 4 secretion, and this was associated with a reduction in STAR and HSD3B protein abundance.Similarly, Annie et al. 20 implied that in prepubertal mice, visfatin inhibits ovarian steroidogenesis.Our study also indicated that visfatin influences steroidogenesis in luteal cells in pigs by regulating the expression of enzymes involved in steroid synthesis.Examination of steroidogenic markers expression on days 10-12 of the estrous cycle showed that visfatin increased the level of STAR, CYP11A1, HSD3B, and CYP19A1.
Prostaglandins have an important function in the proper lifespan of the CL.PGE 2 is a luteotropic factor in many species, including pigs.It maintains P 4 secretion by luteal cells, and its level is highest until the end of the mid-luteal phase.PGF 2α , a luteolytic factor, causes apoptosis of luteal cells, which leads to regression of the CL at the end of the luteal phase 4 .We demonstrated that visfatin (10 and 100 ng/mL) increased PGE 2 secretion almost twofold and threefold on days 2-3 and 10-12 of the estrous cycle, respectively, when the demand for luteal cells is highest.Taking into account these results and the previously mentioned effect on P 4 and E 2 , visfatin can be classified as a luteotropic agent in porcine luteal tissue.There is limited literature on the regulation of prostaglandin levels by visfatin.Gosset et al. 33 demonstrated that in human chondrocytes obtained from patients with osteoarthritis, visfatin excessively stimulated PGE 2 synthesis by increasing the expression of proteins involved in its production.In the context of these studies, the authors concluded that visfatin has a catabolic action in chondrocytes associated with inflammation and it is involved in the pathogenesis of this disease.In the CL, PGE 2 has more protective mode of action.The effect of visfatin on the PGF 2α concentration was noticeable only on days 14-16 of the estrous cycle, when its level was reduced in the presence of all tested visfatin doses.These results could indicate a protective effect of visfatin on luteal cells until the end of the luteal phase in pigs.Interestingly, we observed that FK866 significantly increased the PGF 2α secretion on days 2-3 of the estrous cycle; however, the addition of visfatin completely abolished this effect, restoring the PGF 2α concentration to the control level.This may suggest that visfatin is essential for maintaining the proper level of this prostaglandin.
Prostaglandins act through specific receptors, for PGF 2α it is PTGFR, while PGE 2 may act through several isoforms of the PTGER.In the porcine CL, PTGER isoform 2 (PTGER2) shows the highest expression 34 .Our results regarding the effect of visfatin on the expression of prostaglandin receptors are complex and differ at the transcript and protein levels.Specifically, visfatin increased PTGER2 mRNA expression but decreased its protein expression on days 10-12 of the estrous cycle.Similarly, PTGFR mRNA expression was reduced after treatment with visfatin, while its protein expression was increased.From a functionality standpoint, protein expression provides more crucial information than gene expression.Nevertheless, our results indicate complex regulation at both the transcriptional and translational levels of these proteins, and one of the reasons for the observed changes may be the concentrations of ligands for these prostaglandin receptors.On days 10-12 of the cycle, the PGE 2 level was higher after visfatin treatment.This excess amount of PGE 2 might lead to saturation and desensitization or internalization of the PTGER2 receptor, resulting in a decrease in its protein level 35 .We also noted that visfatin reduced the PGF 2α level at the end of the luteal phase; with the decreased ligand concentration, there could be an increase in receptor expression in this phase to maximize PTGFR availability and to enhance PGF 2α signalling in the regressing CL.This, in turn, could indicate that despite being a luteotropic factor, as we demonstrated in the first experiment, it also supports luteolysis at the proper time, namely the end of the luteal phase.
Visfatin may affect target cells in two ways: it may bind cell surface receptors, such as earlier mentioned TLR4 and CCR5, and activate intracellular signalling pathways, and it may also act as an enzyme, including as an extracellular ectoenzyme 17 .Visfatin/NAMPT is an enzyme involved in the biosynthesis of NAD+, a crucial cofactor in cellular redox reactions.Its availability can impact cellular functions, including those related to steroidogenesis or prostaglandin signalling 36 .We noted that the presence of FK866 in cultures of luteal cells abolished visfatin's effects and promoted the opposite effect of visfatin on most of the observed outcomes, such as P 4 secretion in the early and mid-luteal phases, E 2 level in the early and late luteal phase, expression of steroidogenic enzymes, and prostaglandin secretion.According to the literature data, NAMPT activity is required for visfatin stimulation of steroid secretion in human Gc 18 .Similarly, in prepubertal mice, NAMPT inhibition by FK866 increased E 2 secretion and upregulated the expression of CYP11A1, HSD17B, and CYP19A1 20 .However, our results indicate, that the endocrinological function of the porcine CL is not influenced solely by the enzymatic form of visfatin (as NAMPT).In many cases, the action of visfatin added alone or together with LH or INS was not blocked by FK866.These findings indicate another mechanism beyond NAMPT enzymatic activity through which visfatin acts in luteal cells.Hence, we investigated the possible activation of INSR by visfatin and its impact on the phosphorylation of several protein kinases, and whether they could mediate the action of visfatin in porcine luteal cells.
Prior to our study, researchers had found that visfatin activates the MAPK/ERK1/2 pathway in the ovary; in bovine Gc, it visfatin increased phosphorylation of MAPK3/1 22 , while in hen Gc it reduced phosphorylation of MAPK3/1 21 .Reverchon et al. 18 showed strong activation of ERK1/2, AKT, and p38 upon visfatin treatment in human Gc.In our study, visfatin increased MAPK/ERK1/2, AKT, and AMPK phosphorylation in porcine luteal cells.Furthermore, blocking these kinase signalling pathways revealed their involvement in the stimulatory effect of visfatin on P 4 secretion on days 10-12 of the estrous cycle.In the case of E 2 secretion, there was no difference between the influence of LY294002 alone and LY294002 with visfatin on E 2 level, confirming that AKT is not involved in visfatin-mediated regulation of E 2 secretion.Considering prostaglandins, we focused on examining the involvement of the MAPK/ERK1/2 pathway, due to the fact that this pathway mainly participates in their synthesis.The fact that suppression of visfatin influenced the secretion of both prostaglandins in response to MAPK/ERK1/2 blockade implies that this pathway is involved in visfatin's action.www.nature.com/scientificreports/ The INS-mimetic properties of visfatin exerted by its direct interaction with the INSR are still a subject of discussion.In human osteoblasts 15 and mouse pancreatic beta cells 14 , visfatin stimulated INSR as well as insulin receptor substrate 1 and 2 (IRS-1 and IRS-2, respectively).Our recent studies showed stimulation of INSR phosphorylation in porcine anterior pituitary cells; in addition, INSR mediated the effect of visfatin on gonadotropins secretion 37 .In porcine luteal cells, we noted a reduction of the phosphorylated form of INSR, which is contrary to most reports in the literature 14,15,37 .Nevertheless, blocking the INSR signalling pathway abolished the observed effect of visfatin on the secretion of both steroids and prostaglandins, suggesting that reducing INSR phosphorylation is crucial for visfatin to influence the secretory functions of luteal cells.Considering, the relationship between visfatin and INS observed in our study, we suggest that the effect of visfatin may not be related to the activation of the signalling pathway via INSR itself, but rather to its weakening.On days 10-12 of the estrous cycle, the administration of INS suppressed visfatin-stimulated P 4 secretion.Therefore, blocking INS signalling by decreasing INSR phosphorylation allows visfatin to influence the secretion of steroids.In the human hepatic HEP G2 cell line, Heo et al. 38 also observed that visfatin reduced the levels of phospho-INSR, phospho-IRS-2, phospho-AKT and phospho-glycogen synthase kinase 3 α/β, which are involved in INS signalling processes.
In summary, we showed that visfatin exerts an effect on the endocrine function of porcine luteal cells by influencing the secretion of steroids and prostaglandins and the expression of steroidogenic markers and prostaglandin receptors (Fig. 8).The action of visfatin depends on the phase of the estrous cycle and is modulated by the presence of LH and INS.It exerts its effect not only through the enzymatic activity of NAMPT, but also through the INSR and MAPK/ERK1/2, AKT, and AMPK.Our results indicate that visfatin may be one of important factors regulating of CL physiology in livestock.

Animals and sample collection
The research was carried out on mature cross-breed gilts (Large White × Polish Landrace) at the age of 7-8 months and weighing 140-150 kg from local slaughterhouse in Poland.Samples were collected from pigs intended for commercial purposes and meat processing.The animals were used in accordance with the Act of the 15th of To determine the effect of visfatin on the endocrine function of porcine CL, we conducted a series of in vitro cultures of luteal cells (six independent in vitro cultures of luteal cells, biological replications, n = 6 per group) isolated from CL on days 2-3 (early luteal phase, formation of the CL), days 10-12 (mid-luteal phase, the highest activity of the CL and production of P 4 ), and days 14-16 (late luteal phase, regression of the CL) of the estrous cycle.For each in vitro culture of luteal cells, a subsequent biological replication of the experiment utilized CLs from 5 to 7 pigs.The phases of the estrous cycle were confirmed based on the characteristics of ovarian morphology 39 .Within a few minutes after slaughter, the ovaries were removed, placed in phosphate-buffered saline (PBS, pH 7.4, 4 °C) with a mixture of antibiotics, and transported on ice to the laboratory within 1-1.5 h.

In vitro luteal cell cultures
Luteal cell isolation and the in vitro cell culture were performed by using the same methodology as Rytelewska et al. 40 .In brief, CLs were dissected, minced mechanically, and then enzymatically digested with 0.1% collagenase type V in Hank's Balanced Salt Solution (pH 7.4).We separated small and large luteal cells from CLs and nonsteroidogenic cells, such as endothelial cells.Erythrocytes have been removed during isolation using a special buffer for the red blood cell lysis.The cells were counted after isolation, and their viability was determined with a trypan blue exclusion test.The mean viability of the cells was 95.60% ± 1.54%.Only luteal cells were counted to inoculate the appropriate amount needed for the experiments.The cells were cultured in 6-well culture plates at the final concentration of 2 × 10 6 cells/well (2 mL of medium per well) for western blot analysis, in 24-well plates at a concentration of 2.5 × 10 5 cells/well (1 mL of medium per well) for radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA), and in 96-well plates at a concentration of 9 × 10 4 cells/well (200 µL of medium per well) for transcript levels measured by real-time PCR in a humidified incubator (terms: 37 °C, 95% air, 5% CO 2 ; Binder CB160, DanLab, Poland).Cells were suspended in Ham's F-12 medium enriched with 0.268% sodium bicarbonate, 10% fetal bovine serum, 1% bovine serum albumin (BSA), and a mixture of antibiotics.Next, the cells were precultured for 48 h, and then switched to a fresh medium containing FBS and treated with appropriate hormones/markers.The current study comprised six experiments (Supplementary Table 3).
Experiment 1: In the first experiment, we aimed to determine the basal and LH-and INS-induced effect of visfatin on P 4 and E 2 secretion by the CL during the luteal phase (days 2-3, 10-12, 14-16 of the estrous cycle).Cells were treated with visfatin (cat.no.8424-VF-050, Bio-Techne, Minneapolis, MN, USA) at the dose of 1, 10 and 100 ng/mL, which we chose based on our previous study 37,41 .Moreover, cells were treated with visfatin (10 ng/mL) together with LH (100 ng/mL, from human pituitary, cat.no.L6420, Sigma-Aldrich, St. Louis, MO, USA) or INS (10 ng/mL, from porcine pancreas, cat.no.I5523, Sigma-Aldrich, St. Louis, MO, USA) or with LH (100 ng/mL) and INS (10 ng/mL).Doses of LH we chosen based on a previous paper Kurowska et al. 7 , while the INS concentration was based on the study by Gavin et al. 42 .Additionally, we used FK866 (10 nM, cat.no.F8557, Sigma-Aldrich, St. Louis, MO, USA) with all mentioned hormones; the dose of FK866 we established based on the study by Reverchon et al. 18 .Importantly, FK866 was added already during the seeding of luteal cells, and changing the medium to fresh with the tested hormones.After incubation for 24 h, the culture medium was collected and stored in − 20 °C for further measurement of P 4 with RIA and E 2 with ELISA.
Experiment 2: We chose one phase of the estrous cycle (days 10-12) to evaluate whether visfatin regulates steroid synthesis by influencing the expression of markers involved in this process in the CL.Due to the fact that the production of steroids, especially P 4 , is the highest in mid-luteal phase, we decided to check the expression of steroidogenic factors on days 10-12 of the estrous cycle.Luteal cells were treated with visfatin (1-100 ng/mL) and the FK866 blocker or with visfatin at the dose of 10 ng/mL.After incubation for 24 h, cell lysates were collected to examine STAR, CYP11A1, HSD3B, and CYP19A1 mRNA and protein expression by real-time PCR and western blot, respectively.
Experiment 3: In this experiment, we examined the effect of visfatin on the secretion of prostaglandins by luteal cells during the entire luteal phase (days 2-3, 10-12, and 14-16 of the estrous cycle).Cells were treated with visfatin (1-100 ng/mL) and FK866 alone or with visfatin at the dose of 10 ng/mL.Following incubation, medium was collected to measure the PGE 2 and PGF 2α levels with commercially available ELISA kits.
Experiment 4: Based on the results obtained from Experiment 3, we selected one phase of the estrous cycle where visfatin exerts an effect on prostaglandins to check the expression of prostaglandin receptors.Thus, we examined PTGER2 expression on days 10-12 and PTGFR expression on days 14-16 of the estrous cycle.Luteal cells were stimulated with visfatin (1-100 ng/mL) as well as the FK866 alone or with visfatin at the dose of 10 ng/ mL.After incubation for 24 h, cell lysates were collected to examine PTGER2 and PTGFR mRNA and protein expression with real-time PCR and western blot, respectively.
Experiment 5: We determined the visfatin effect on the activation of INSR and several intracellular signalling pathways in the CL on days 10-12 of the estrous cycle.For this purpose, luteal cells were incubated with visfatin at the dose of 10 ng/mL for 2, 5, 15, and 30 min.Then, medium was collected to examine the phosphorylated and total forms of INSR by ELISA, and cell lysates were collected to examine protein expression of the phosphorylated and total forms of MAPK/ERK1/2, AKT, and AMPK using western blot.
Experiment 6: In the final experiment, we evaluated the involvement of INSR and MAPK/ERK1/2, AKT, and AMPK in visfatin's action on steroid and prostaglandin secretion.We selected only one stage of the estrous cycle based on results obtained in Experiments 1 and 3; P 4 , E 2, and PGE 2 secretion was determined on days 10-12, and PGF 2α secretion was determined on days 14-16 of the estrous cycle.Cells were treated with the pharmacological blockers of INSR (S961, 1 µM), AKT (LY294002, 20 µM), MAPK/ERK1/2 (U0126, 10 µM), or AMPK www.nature.com/scientificreports/(Dorsomorphin, 10 µM).We chose the doses of these blockers based on the studies by Elliot et al. 43 , Zhao et al. 44 , and Reverchon et al. 18 for S961, LY294002, U0126, and Dorsomorphin, respectively.Luteal cells were preincubated with blockers for 1 h and then we added visfatin at the concentration of 10 ng/mL.After incubation for 24 h, culture medium was collected to measure P 4 secretion with RIA, and E 2 , PGE 2 , and PGF 2α secretion with ELISA.

RIA
The concentrations of P 4 in the culture media were determined by RIA with tritium labelling ( 3 H, a source of β-radiation) according to the method described by Ciereszko et al. 45 .The specificity of the antibodies against P 4 (SO/91/4) was previously reported 45 .Before the main analysis, a preliminary test was performed to determine the optimal dilutions of the anti-P 4 antibody and culture medium to ensure the most effective detection range for the assay.As a result, an antibody dilution of 1:3000 and a culture medium sample dilution of 1:500 were used.The probe radioactivity levels were measured using a Hidex 300 SL scintillation counter (Hidex Oy, Turku, Finland) with the Microwin 2000 software (Mikrotek Laborsysteme GmbH, Overath, Germany).All standards were run in triplicate and all culture medium samples were run in duplicate.The mean antibody binding was 27.55% ± 2.38%.The sensitivity of the assay was 1 pg/mL, while the range of the standard curve was 1-1000 pg/ mL.The P 4 concentration in each sample was determined from the standard curves, which were plotted as polynomial trend lines representing the count per minute (CPM) values standardized against a blank probe for each standard solution versus their respective concentrations.The intra-and inter-assay coefficients of variation were 3.98% ± 0.99% and 9.63% ± 2.02%, respectively.

ELISA
The concentrations of E 2 , PGE 2, and PGF 2α as well as phospho-INSR and total-INSR in culture media were determined by using commercially available ELISA kits according to the manufacturers' protocols.Supplementary Table 4 provides more details about the assays used in this study.The samples were run in duplicate within the same assay.Absorbance values were measured at 450 nm using a Varioskan LU Multimode Microplate Reader and the SkanIt Software 6.1.1 (Thermo Fisher Scientific, MA, USA).

Real-time PCR
TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA, USA) were employed to quantify the mRNA expression of markers implicated in steroid synthesis, as well as prostaglandin receptors.Total RNA isolation and cDNA synthesis were performed according to the TaqMan Gene Expression Cells-to-CT Kit protocol (cat.no.AM1728, Applied Biosystems, Carlsbad, CA, USA).The RNA and cDNA concentrations were determined based on optical density at 260 and 280 nm.Amplifications were executed using the StepOnePlus system (Applied Biosystems, Carlsbad, CA, USA) under the manufacturer's instructions, utilizing TaqMan-specific primers for STAR (assay ID: Ss03381250_u1), CYP11A1 (assay ID: Ss03384849_u1), HSD3B (assay ID: Ss03391752_m1), CYP19A1 (assay ID: Ss03384876_u1), PTGER2 (assay ID: Ss03374177_g1), and PTGFR (assay ID: Ss03393819_s1).The final 20-µL reaction volume comprised the TaqMan Gene Expression Master Mix and 50 ng of cDNA.Relative gene expression was normalized against the reference gene PPIA 7,46 (assay ID: Ss03394782_g1).The relative gene expression levels were determined by following the method outlined by Livak and Schmittgen 47 , using the comparative cycle threshold (2 -ΔΔCt ) approach.

Western blot
Luteal cells were lysed using Tissue Protein Extraction Reagent (cat.no.78510, Thermo Fisher Scientific, MA, USA) with addition of protease and phosphatase inhibitors.Equal quantities of lysates (30 μg protein/sample) with Laemmli buffer (cat.no.23225, Sigma-Aldrich, MO, USA) were denatured at 95 °C for 5 min, then separated in 10% sodium dodecyl sulphate-polyacrylamide gels, and transferred onto polyvinylidene fluoride membranes (cat.no.IPVH00010, Sigma-Aldrich, MO, USA).The membranes were incubated in 0.02 M Tris-buffered saline with Tween 20 (TBST) containing 5% BSA for 1 h at 20-22 °C to block nonspecific protein binding.Subsequently, the membranes were incubated overnight at 4 °C with primary antibody.Supplementary Table 5 provides detailed information about antibodies used for this method.The next day, following TBST washes, the membranes were incubated with horseradish peroxidase-conjugated antibody (diluted at 1:1000) (Supplementary Table 5) for 1 h at 20-22 °C.Chemiluminescence detection, using the Immobilon Western Chemiluminescent HRP Substrate (cat.no.WBKLS0500, Sigma-Aldrich, MO, USA), revealed signals that were visualized using the ChemiDoc™ imagining system (Bio-Rad, CA, USA).Actin served as a loading control.The ImageJ software (US National Institutes of Health, Bethesda, MD, USA) was used for densitometric analysis of the protein bands.Representative blots are included as Supplementary Fig. 1.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA, USA).All experimental data are presented as mean ± standard error of the mean of experiments which were performed in six replicates (n = 6).Extreme values were rejected according to the three-sigma rule 48 .The data were evaluated to determine whether they met the assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) and then analysed with one-way ANOVA followed by Tukey's test.Statistically significant differences (p < 0.05) are indicated by different letters (Supplementary Table 6).Additionally, we performed two-way ANOVA to examine differences in steroid secretion levels with two independent variables (main factors): VIS and FK866, and the interaction of those factors, i.e.VIS*FK866 for each experimental setup and phase of the estrous cycle (Supplementary Tables 1 and 2).

Figure 1 .
Figure 1.The effect of visfatin (VIS) on basal and luteinizing hormone (LH)-and insulin (INS)-induced progesterone (P 4 ) secretion by luteal cells collected from CL on days 2-3 (A), 10-12 (C), and 14-16 (E) of the estrous cycle.Luteal cells were also treated with the blocker FK866 and tested for the effect on P 4 secretion (B, D, F).The data are presented as the mean ± standard error of the mean (n = 6 replicates).Dots marked individual values indicating the distribution of a range of values.Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).

Figure 2 .
Figure 2. The effect of visfatin (VIS) on basal and luteinizing hormone (LH)-and insulin (INS)-induced estradiol (E 2 ) secretion by luteal cells collected from CL on days 2-3 (A), 10-12 (C), and 14-16 (E) of the estrous cycle.Luteal cells were also treated with the blocker FK866 and tested for the effect on E 2 secretion (B, D, F).The data are presented as the mean ± standard error of the mean (n = 6 replicates).Dots marked individual values indicating the distribution of a range of values.Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).

Figure 3 .
Figure 3.The effect of visfatin (VIS) and its blocker, FK866, on mRNA and protein abundance of steroidogenic markers: steroidogenic acute regulatory protein (STAR) (A, B), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) (C, D) hydroxy-delta-5-steroid dehydrogenase (HSD3B) (E, F), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) (G, H) in porcine luteal cells isolated on days 10-12 of the estrous cycle.Abundance of mRNA was expressed as a fold of controls.The protein abundance was analyzed by western blot.The results are shown as representative immunoblots and a bar graph with densitometry measurement of relative target protein content normalized to actin.The data are presented as the mean ± standard error of the mean (n = 6 replicates).Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).

Figure 4 .
Figure 4.The effect of visfatin (VIS) and its blocker, FK866, on prostaglandin E 2 (PGE 2 ) (A-C) and prostaglandin F 2α (PGF 2α ) (D-F) secretion by luteal cells collected from CL on days 2-3, 10-12, 14-16 of the estrous cycle.The data are presented as the mean ± standard error of the mean (n = 6 replicates).Dots marked individual values indicating the distribution of a range of values.Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).

Figure 5 .
Figure 5.The effect of visfatin (VIS) and its blocker, FK866, on mRNA and protein abundance of the prostaglandin E 2 receptor (PTGER2) (A and B) and the prostaglandin F 2α receptor (PTGFR) (C and D) in porcine luteal cells isolated on days 10-12 and 14-16 of the estrous cycle, respectively.Abundance of mRNA was expressed as a fold of controls.The protein abundance was analysed by western blot.The results are shown as representative immunoblots and a bar graph with densitometry measurement of relative target protein content normalized to actin.The data are presented as the mean ± standard error of the mean (n = 6 replicates).Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).

Figure 6 .
Figure 6.The time-dependent effect of visfatin at the dose of 10 ng/mL on the concentration of phosphorylated and total insulin receptor (INSR) (A), and the protein abundance of phosphorylated and total form of extracellular signal-regulated kinase 1/2 (ERK1/2) (B), protein kinase B (AKT) (C), and 5′AMP-activated protein kinase (AMPK) (D) in porcine luteal cells isolated on days 10-12 of the estrous cycle.The concentration of phosphorylated and total INSR in culture medium was measured using enzyme-linked immunosorbent assay.The protein abundance was determined by western blot method.The results are shown as representative immunoblots and a bar graph with densitometry measurement of the phosphorylated form relative to the total form.The data are presented as the mean ± standard error of the mean (n = 6 replicates).Within each panel, bars/means without a common capital letter differs significantly (p < 0.05).